Modified nucleosides have been used as therapeutic agents, diagnostic agents, and for incorporation into oligonucleotides to improve their properties (e.g., stability).
SELEX (Systematic Evolution of Ligands for EXponential Enrichment) is a method for identifying oligonucleotides (referred to as “aptamers”) that selectively bind target molecules. The SELEX process is described in U.S. Pat. No. 5,270,163, the contents of which are herein incorporated by reference in their entirety. The SELEX method involves the selection and identification of oligonucleotides from a random mixture of oligonucleotides to achieve virtually any desired criterion of binding affinity and selectivity. By introducing specific types of modified nucleosides to the oligonucleotides identified in the course of the SELEX process, the nuclease stability, net charge, hydrophilicity or lipophilicity may be altered to provide differences in the three dimensional structure and target binding capabilities of the oligonucleotides. Thus, different modified nucleosides provide the ability to “tune” the desired properties of an oligonucleotide selected in the course of SELEX.
Modified deoxyuridine nucleotides, bearing an N-substituted-carboxamide group at the 5-position, have proven to be valuable tools for improving in vitro selection of protein-binding aptamers (SELEX process) (see, e.g., Gold et al., 2010; Hollenstein, 2012; and Imaizumi et al., 2013) and for post-SELEX optimization of binding and pharmacokinetic properties of the selected aptamers (see, e.g., Davies, et al., 2012; Lee et al., 2010; Kerr et al., 2000; and Gaballah et al., 2002). The general synthesis of uridine-5-carboxamides relied on a common activated ester intermediate, 5-(2,2,2-trifluoroethoxycarbonyl)-2′-deoxyuridine (1), which was originally reported by Matsuda and coworkers (see, e.g. Nomura et al., 1997). Treatment of this activated ester with various primary amines (1.2 eq., 60° C., 4 h) affords the corresponding 5-N-substituted-carboxamides). Matsuda also disclosed the analogous activated ester in the cytidine series, N-acetyl-5-(2,2,2-trifluoroethoxycarbonyl)-2′-deoxycytidine (see, e.g. Nomura et al., 1996). However, this intermediate was less practically useful for synthesis of cytidine-5-carboxamides due to the lability of the N-acetyl protecting group and the instability of the N-acetyl-5-iodo-cytidine synthetic precursors.
There continues to be a need for alternative composition for improving oligonucleotide target binding agents, and further methods for synthesizing such compositions. The present disclosure meets such needs by providing novel cytidine-5-carboxamide modified compositions.